lane tracking

>Our lab is spending far too much time tracking lanes. We retrack more
>than half our gels. Hence they want me write lane tracking software,
>which I know won't be easy or short.
>>ABI's lane tracking software does fine when the lanes run straight.
>In a previous job, I remember that some changes were made in the
>chemistry or gel preparation protocol that made things run straight.
>It wasn't 100%, but it helped a lot.
>>Unfortunately, (`cause I'm the computer guy) I don't remember
>what was done. Can anybody give me some ideas on what to try?
>>(We run ABI 377's with a 32 well comb.)
>> Thanks,
> Ron L.
>>--
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>* RON LUNDSTROM ron at eri.uchsc.edu *
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Dear Ron,
We also had a lot of trouble initially with the lanes curving inwards at
the beginning of the sequence ladders. This was clearly due to salt in the
samples, as it got worse when we added salt. The spin columns we were using
came in 10 mM TE and the instructions did not include a prewash step. This
is a problem since after collection of the eluate it is necessary to vacumn
concentrate to dryness and then rehydrate in a smaller volume - thus
raising the salt concentration. The solution was to prewash the desalting
columns used for removal of the dye terminators with distilled water prior
to sample loading. The result was absolutely straight lanes.
Hope this helps,
-David
David F. Bishop, Ph.D. | EMail:bishop at msvax.mssm.edu
Dept. of Human Genetics, Box 1203 |
Mount Sinai School of Medicine | Phone: (212) 241-6946
5th Avenue and 100th Street, NY,NY 10029 | FAX: (212) 360-1809